Many instruments have been developed to measure the quantity of analytes in various biological samples, for example urine, blood, salvia, or extracts of mucus or tissue. Typically, a sample liquid is applied to a surface containing reagents that react with the analyte. The reagents produce a detectable response that is measured and related to the amount of the analyte. The surface can be hydrophilic or hydrophobic in nature, e.g. filter paper compared to polystyrene. Some devices use combinations, such as urinalysis strip tests that use hydrophilic filter paper pads on top of a hydrophobic polystyrene handle. In the typical test, a strip containing reagents is dipped, i.e. fully immersed in a liquid sample, and the reaction between the analyte in the sample and the reagents is measured, typically by optical methods. Other devices include microchips that use hydrophilic substrates connected to capillaries molded of polystyrene. The reagents themselves can be water soluble or insoluble and dried onto the supporting surface, as in test strips. Or, they could be added as a liquid to a microchip. Additional liquid reagents can be applied to the surfaces already containing dried reagents. Typically this application occurs after a sample has been applied. The sample volume should be as small as possible for obvious reasons relating to cost and convenience. What is less obvious is that it is often difficult to obtain uniform and accurate responses when applying small amounts of liquid reagents or biological samples to surfaces containing reagents.
Most biological samples and liquid reagents will have a significant water content and thus will be compatible with hydrophilic substrates and incompatible with hydrophobic surfaces. The samples and reagent liquids when dispensed spread rapidly across hydrophilic substrates and are repelled by hydrophobic substrates. The contact between the dispensed liquid and the reagents on the surface can be made by capillary action or directly. However, when substrates are relatively hydrophobic, the dispensed liquid will form beads on the surface of the substrate that attempt to minimize their contact with the surface. Dispensed liquids therefore do not spread uniformly over the reagent. Another difficulty associated with dispensing liquids is that the dried reagents may be either water soluble or water insoluble in nature. The insoluble dry reagents may not be readily accessible to the liquid samples, or soluble reagents may be dissolved and move with the liquid on the substrate. The reagents ideally should contact the sample uniformly since the measurable response of the reagents to the sample, e.g. color development, should be uniform in order to obtain an accurate reading of the analyte in the sample.
Another problem related to obtaining good contact between a dispensed liquid and a reagent on a surface is related to the physical nature of the samples. They vary in their physical properties such as surface tension, viscosity, total solids content, particle size and adhesion. Therefore they are not easily deposited in consistent volumes uniformly over the reagent-covered substrate. Also, as the amount of the liquid sample is reduced, it becomes increasingly difficult to apply a consistent amount of a sample with varying properties to the reagents. In contrast, ink-jet printing and the like rely on liquids developed for such uses and having consistent physical properties.
Deposition of droplets of liquid is a familiar operation. Examples include the ink jet-printer, either piezoelectric or bubble actuated, which forms print from the controlled deposition of multiple small droplets of about 2 to 300 μm diameter (typically 50 μm) containing from a few femtoliters to tens of nanoliters. Other methods of depositing small droplets have been proposed, which generally employ piezoelectric principles to create droplets, although they differ from typical ink-jet printers. Examples are found in U.S. Pat. Nos. 5,063,396; 5,518,179; 6,394,363; and 6,656,432. Deposition of droplets of larger droplets through syringe type pipette is known to be reproducible in diagnostic systems from 3 to 100 μL. This corresponds to single droplet diameters of about 2 to 6 mm. A commercial example of such pipette systems is the CLINITEK ALTAS® urinalysis analyzer. The droplet size can be greater or less than the nozzle size depending on the nozzle shape, pump type and pressures applied.
The problems discussed above are particularly observed when a liquid sample is dispensed as droplets onto a reagent-containing pad. The applicants found that the interactions of the pad's surface and the reagents were creating inaccurate responses when the sample was added as a droplet, rather than completely covering the reagent pad by immersing the reagent pad (dipping it) into the sample liquid, as is frequently done. Large droplets on the order of 3 to 100 μL do not transfer into the reagent when the substrate is too hydrophobic and form a bubble on the surface. They overwhelm the reagent with excess fluid if the surface is hydrophilic. Smaller droplets, of a few femtoliters to tens of nanoliters, can also be a problem when deposited on a substrate that is too hydrophobic as they lack the volume to completely cover the surface area and will randomly aggregate in non-uniform patterns. Small drops also allow open spaces for migration of water-soluble reagents. These tiny droplets are also prone to evaporation of liquids and to formation of aerosols, which are considered to be bio hazardous if comprised of urine or blood specimens. Thus, if a liquid is to be deposited as droplets on test pads, rather than dipping the pads in the sample, improvements are needed.
After contact between dispensed liquids and reagents is complete, the results may be read using one of several methods. Optical methods are commonly used, which rely on spectrographic signals to produce responses. Results must be reproducible to be useful. Optical measurements are affected by the reagent area viewed and by the time allowed for the dispensed liquids and reagents to react. Formation of non-uniform areas within the field of view and changes in the amount of reaction time needed increase errors. For example, a measurement made of a sample or reagent which has spread non-uniformly across the substrate gives a different result each time it is read.
It is always an objective of those who develop and improve methods of analyzing biological samples to provide accurate and consistent results. The present inventors propose new methods for improvement in the results obtained when liquid biological samples or liquid reagents are deposited on surfaces containing dried reagents, particularly when the sample is deposited as droplets.